During the first day that embryonic amphibian spinal neurons are electrically excitable, their electrophysiological properties undergo a stereotyped change. The potassium current density increases 3-fold, causing the duration of the calcium-dependent action potential and consequent calcium influx to decrease. Does this result from transcriptional, translational or post-translational regulation of potassium channel gene expression? The proposed studies address this question by determining the normal levels of potassium channel RNA and protein and then perturbing these levels to examine the functional consequences for the differentiation of excitability. Two Xenopus neuronal potassium channel genes have been cloned and will be used to achieve the specific molecular perturbations. Four aims outline the research plan: 1. How are potassium channel RNA and protein levels regulated in developing embryonic neurons? 2. Do altered potassium channel RNA levels disrupt the development of excitability? 3. Are translational mechanisms involved in regulating potassium channel function? 4. What are the consequences of excess potassium channel RNA on neuronal development? The experimental approach takes advantage of the accessibility of the Xenopus embryo for molecular manipulations and analyses of mechanisms of neuronal development. The techniques include standard RNA (Northerns, RNAase protection, in situ hybridization) and protein methods (generation of fusion proteins and antisera, Westerns, immunocytochemistry), injections of nucleic acids into cleavage stage Xenopus embryos, synthesis of RNA in vitro, mutagenesis of DNA (insertional and site-directed), whole cell voltage and current clamp of neurons developing in vitro and in situ, two electrode voltage clamp recording of currents induced in oocytes by functional expression of cloned genes, and examination of spinal cord organization immunocytochemically in whole mount preparations of embryos. Functional regulation of potassium currents in developing neurons is pivotal for the well-known changes in action potential duration and primary ionic dependence. The immediate goal is to elucidate the molecular mechanisms involved in regulating potassium channel function in developing neurons. These studies will provide information towards the long-term goal of understanding the functional role of developmentally regulated potassium currents and action potentials in the emerging vertebrate nervous system. Preliminary data indicate that elevated potassium channel RNA levels are incompatible with later aspects of neuronal differentiation, and the underlying mechanisms will be identified. This information will be valuable for evaluation and eventual treatment of alterations in excitability associated with disorders of the developing nervous system such as epilepsy or trisomy 21.